Cholera epidemics occur seasonally in areas such as Bangladesh, and outbreaks can also strike in vulnerable regions, as has occurred recently in Haiti. The disease is caused by Vibrio cholerae, a water-borne bacterium that colonizes the small intestine, and its symptoms include severe diarrhea and vomiting which can lead to death if the patient is not treated promptly.

Lytic phages are viruses that specifically attack and kill bacteria. After replicating many times inside the bacterial cell, the phages break open and destroy the cell. Over time a bacterial population can evolve to resist this phage ‘predation’; however, it is not known if bacterial pathogens need to defend themselves against phage attack when they infect humans. It had been suggested that phages might affect the progress of cholera infections in people, but molecular evidence that supports this hypothesis was lacking.

When testing stool samples from Haitian cholera patients, Seed et al. found one sample contained a lot of lytic phage relative to the amount of V. cholerae present. This phage was very similar to—but distinct from—a phage found in Bangladeshi patients.

The V. cholerae bacteria isolated from the stool sample were resistant to attack by the phage. Sequencing the genome of individual bacteria from this sample revealed that each had a mutation that made them resistant to the phage; and while many types of these mutations were found, these were the only differences between all the V. cholerae bacteria in this patient sample. This suggests that this resistance developed independently many different times within the patient due to strong selective pressure from phage predation.

When Seed et al. looked at a phage-positive stool sample from a Bangladeshi patient, more mutations that made the bacteria resistant to this phage were found; however, these mutations were different again from the ones in the Haitian bacteria. Because of the nature of these mutations the bacteria from this patient were rendered unable to cause disease and non-transmissible.

This work shows that phages can indeed have access to pathogenic bacteria during human infection. It also indicates that the pressure imposed by phage predation can, in some cases, be so strong that the bacteria lose their virulence and ability to spread to other humans in order to become resistant to the phage.

Traditional views of host–pathogen interactions are of a battle between two opposing organisms. This perspective is being challenged with greater appreciation of the influence of the host's microbial ecosystem on these interactions (Lozupone et al., 2012). Bacteriophages influence bacterial populations in many ecosystems and specifically temperate phages play a role in many diseases through lysogenic conversion (Brüssow et al., 2004). In contrast, the impact of lytic phage predation on bacterial pathogens in the context of human disease is under-appreciated. Vibrio cholerae is a water-borne pathogen responsible for the diarrheal disease cholera. In order for V. cholerae to colonize the human small intestine and elicit diarrhea, it produces a set of virulence determinants including cholera toxin (Herrington et al., 1988). The ToxR signaling cascade activates expression of these virulence factors in response to host environmental stimuli (Miller et al., 1987; Taylor et al., 1987; DiRita et al., 1991; Childers and Klose, 2007). Cholera epidemics appear with regular seasonality in regions like Bangladesh (Faruque et al., 2005) and can arise unpredictably in vulnerable regions as exemplified by the epidemic that recently began in Haiti following the single-source introduction of a pandemic V. cholerae O1 strain from another continent (Chin et al., 2011; Cravioto et al., 2011; Frerichs et al., 2012; Katz et al., 2013). Lytic phages are hypothesized to impact cholera disease burden in Bangladesh (Faruque et al., 2005). The predation of V. cholerae by phages following their co-ingestion from the environment is central to this hypothesis; however, molecular evidence in support of this hypothesis is lacking.

We recently described the ICP2 species of V. cholerae-specific, virulent podoviruses that are found sporadically in cholera patient stool samples collected since 2001 in Bangladesh (Seed et al., 2011 and present study). To begin to address the geographic diversity of cholera phages in patient stools, we tested by plaque assay for the presence of phages within nine Haitian cholera patient stool samples collected in 2013. We identified one sample that had a high titer of phage (10⁸ PFU/ml) relative to V. cholerae (10⁵ CFU/ml). Whole genome sequencing and comparative analysis of this phage, named ICP2_2013_A_Haiti, revealed 84% identity over 93% of its genome to ICP2_2011_A, a phage isolated from Bangladesh in 2011. An alignment of the annotated ICP2 genome (Seed et al., 2011) with the three available ICP2-related isolates shows that synteny is conserved between these geographically distinct phages, with no significant genome rearrangements observed (Figure 1—figure supplement 1). The Haitian ICP2 isolate is strikingly similar, although clearly distinct, from Bangladeshi ICP2 isolates, and is the first lytic phage reported to be associated with epidemic V. cholerae in Haiti.

Remarkably, we observed that most V. cholerae isolates recovered from the same stool sample as ICP2_2013_A_Haiti were resistant to infection by this phage. We investigated phenotypic heterogeneity within this stool sample by testing 269 single colony isolates for sensitivity to ICP2_2013_A_Haiti and found that 267 (>99%) were phage-resistant. Eight phage-resistant isolates and the two phage-sensitive isolates from this sample were subjected to whole genome sequencing. All 10 isolates were isogenic except for mutations within ompU encoding the major outer membrane porin (Supplementary file 1). Furthermore, the ompU mutations in the eight phage-resistant isolates were heterogeneous. We sequenced ompU from an additional 11 phage-resistant isolates from this stool sample and found a total of six unique ompU alleles (Figure 1A). When each of these alleles was used to replace ompU in a clean genetic background, all conferred resistance to ICP2, yet produced normal amounts of OmpU (Figure 2 and data not shown), suggesting that ICP2 uses OmpU as a receptor to initiate infection and that the mutations disrupt this interaction. These data showing that intra-host V. cholerae isolates are isogenic, only differing in ICP2 resistance mutations, indicates that they diverged within the patient. Furthermore, the presence of multiple different ICP2 resistance mutations within this single host suggests that selection of phage resistance occurred multiple, independent times during the course of infection. We also found evidence for ICP2-mediated selection of OmpU mutants in isolates from Bangladesh. Analysis of the ompU sequence from 54 clinical isolates collected between 2001 and 2011 showed that 15% had non-synonomous mutations (Figure 1A, Figure 1—figure supplement 2) and these alleles were sufficient for ICP2 resistance (Figure 2). One of the OmpU mutants, G325D, was observed in clinical isolates from Haiti and Bangladesh (Figure 1A). Interestingly, seven of the eight mutant OmpU proteins detected in the Haitian and Bangladeshi clinical isolates had alterations in two predicted extracellular loops, while the eighth had an altered transmembrane segment adjacent to the first of these extracellular loops (Figure 1B). This suggests that ICP2 interacts specifically with these exposed loops to initiate infection.

Figure 1 with 2 supplements see all

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Figure 2

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We also tested for V. cholerae population heterogeneity within an ICP2-positive stool sample isolated in Bangladesh in 2011. Of 46 single colony isolates tested for sensitivity to ICP2_2011_A, 22% were resistant to the phage. Whole genome sequencing of two phage-sensitive and four phage-resistant isolates from this sample revealed that the strains were isogenic, except that phage-resistant isolates had nonsense mutations in toxR, the direct transcriptional activator of ompU (Crawford et al., 1998; Supplementary file 2). We sequenced toxR from an additional eight phage-resistant isolates from this stool sample and found that, analogous to what was observed in the Haitian patient sample, there were multiple unique toxR alleles, which included two non-synonomous and two nonsense mutations (Figure 1C). Each of the four mutant toxR alleles conferred ICP2 resistance and abrogated expression of OmpU when used to replace wild-type toxR in a clean genetic background (Figure 2). Both ICP2-sensitivity and OmpU expression were restored to the clinical toxR mutants by expressing ompU in trans or by reverting the toxR allele to wild-type (data not shown), indicating that these mutant toxR alleles are necessary and sufficient for ICP2 resistance and that this resistance is mediated through loss of OmpU expression.

To address the potential consequences of the phage-resistance mutations on V. cholerae fitness, we tested the four most frequently isolated ompU alleles and all four toxR alleles in survival and growth assays. Recent Tn-seq analyses of V. cholerae showed that OmpU is critical for fitness in an infant rabbit infection model (Fu et al., 2013; Kamp et al., 2013) and for dissemination from the host into pond water (Kamp et al., 2013). OmpU has also been shown to be important for protection against the bactericidal effect of bile salts (Provenzano et al., 2001), cationic peptides (Mathur and Waldor, 2004), and intestinal organic acids (Merrell et al., 2001). When we tested for survival in bile and for competitive fitness in pond water we found that the four OmpU mutants are fully fit (Figure 3A,B), consistent with their wild-type levels of OmpU expression (Figure 2). Moreover, the OmpU(G325D) mutant was fully virulent in a single round, competitive infection in infant rabbits (Figure 4, first column). However, the OmpU mutants, particularly the A195T and G325D mutants, showed a mild competitive defect after multiple passaging in growth medium (Figure 3C). The mild growth defect of the OmpU mutants in conjunction with the low prevalence of ICP2 (Seed et al., 2011) may explain why the ICP2-resistant ompU variants do not become fixed in the population (Figure 1—figure supplement 2). In sharp contrast, in the presence of ICP2 we observed a 10,000-fold enrichment of the OmpU(G325D) mutant over the wild-type after infection of infant rabbits (Figure 4), indicating that strong selective pressure is imposed by phage predation during V. cholerae infection. This emulates what we hypothesize happens in human infections in the presence of ICP2 and is the first demonstration of phage predation of V. cholerae in the context of a diarrheal disease model.

Figure 3 with 1 supplement see all

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Figure 4

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V. cholerae strains harboring any of the four ToxR mutations were avirulent in the infant mouse model of infection and were indistinguishable from a ΔtoxR mutant (Figure 3D). The clinical isolates containing these mutations were also at least 100-fold attenuated (Figure 3D—figure supplement 1), indicating that these isolates do not harbor compensatory mutations. The cytoplasmic amino terminus (D17-E112) of ToxR where the two non-synonomous mutations mapped has homology to the winged helix-turn-helix family of transcription activators and is involved in DNA binding and transcriptional activation (Miller et al., 1987; Ottemann et al., 1992). A number of point mutations in this domain were previously shown to abrogate activation of ompU and virulence genes (Ottemann et al., 1992; Morgan et al., 2011). The severe virulence defects of the two non-synonomous toxR mutants from the Bangladeshi patient sample (Figure 3D) are consistent with an inability of the encoded mutant ToxR proteins to activate these genes. These toxR mutants would not colonize the human small intestine and be recovered in the secretory diarrhea if ingested (Herrington et al., 1988), which further supports our conclusion that these mutants arose due to ICP2 predation during cholera infection in this individual. This also indicates that, in this patient, predation and selection of phage-resistant mutants likely occurred late in infection when colonization and virulence gene expression were no longer required for the development of acute symptoms (Merrell et al., 2002).

We have shown that lytic phages are an unexpected ‘third party’ that impose significant bactericidal pressure on an environmentally transmitted pathogen during the natural course of infection in humans. We observed that adaptations to phage predation involve tradeoffs in evolutionary fitness and provide a molecular mechanism for phage predation impacting V. cholerae transmission and seeding of environmental reservoirs. Our results highlight that host–pathogen interactions are embedded within and strongly affected by a complex microbial ecosystem. Predator-prey dynamics are notably absent between microbiotal phage and their bacterial hosts in the intestinal ecosystem in healthy humans (Reyes et al., 2010). In contrast, our findings indicate that diseased states, which are often accompanied by significant bacterial proliferation, likely facilitate these predatory interactions.

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Thank you for sending your work entitled “Evolutionary Consequences of Intra-Patient Phage Predation on Microbial Populations” for consideration at eLife. Your article has been favorably evaluated by Richard Losick (Senior editor), a Reviewing editor and 2 reviewers, one of whom, Marie-Agnès Petit, has agreed to reveal her identity.

The Reviewing editor and the two reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

Seed et al. report a very clear cut and elegant set of data providing evidence of a natural, in vivo predator prey relationship between Vibrio cholerae and ICP2-like virulent phages of the podoviridae family, upon human infection. In two patients infected with V. cholerae, and having high loads of ICP2 phage, they discovered two different panels of Vibrio mutations, in ompU and toxR, resulting in the mutation or repression of the OmpU receptor, respectively. They also establish that in a collection of 54 isolates of V. cholerae, 15% had an ompU mutation.

To our knowledge, this is the first report of a predator-prey evolution taking place in the human intestine. In addition, the toxR mutants have attenuated virulence, suggesting that the presence of the virulent phage may have contributed to the elimination of the infection by natural evolution. This is not true however for the ompU mutants that appear almost not attenuated in virulence/ survival phenotypes. The contrast between the two sets of mutants isolated is striking. As suggested by the authors, such an evolution, not reported until now for bacterial strains in healthy microbiota, may have taken place due to the disequilibrium of the resident microbiota in patients. This is a new concept, and the study certainly deserves publication.

This study also represents an extremely interesting and timely topic in light of the recent phage therapy re-birth. One of the reviewers wrote: “I was amazed by the results on ompU mutant selection by phage co-infection in these conditions and was surprised that this selective effect had never been observed in the cholerae OmpU. I went to Genbank and retrieved the various cholerae OmpU (VC0633) sequences and compared its variation to its neighboring D-alanyl-D-alanine carboxypeptidase (VC0632). I did not perform a statistical analysis, but it is obvious that OmpU is more variable than VC0632. However, this can have many different causes, but strikingly, most of the OmpU variants carry mutation(s) in the very same positions as those identified in this study (A182, VG 324/5...). A simple but thorough analysis of the OmpU mutations could add more weight to the conclusions presented in this manuscript on the selective force exerted by phage over the time.”

[…] This study also represents an extremely interesting and timely topic in light of the recent phage therapy re-birth. One of the reviewers wrote: “I was amazed by the results on ompU mutant selection by phage co-infection in these conditions and was surprised that this selective effect had never been observed in the cholerae OmpU. I went to Genbank and retrieved the various cholerae OmpU (VC0633) sequences and compared its variation to its neighboring D-alanyl-D-alanine carboxypeptidase (VC0632). I did not perform a statistical analysis, but it is obvious that OmpU is more variable than VC0632. However, this can have many different causes, but strikingly, most of the OmpU variants carry mutation(s) in the very same positions as those identified in this study (A182, VG 324/5...). A simple but thorough analysis of the OmpU mutations could add more weight to the conclusions presented in this manuscript on the selective force exerted by phage over the time.”

We thank the reviewers for their comments and idea to include more OmpU sequences. We used BLASTP to identify OmpU in other Vibrio cholerae isolates in GenBank. Homologues were aligned and WebLogo (Crooks et al., 2004) was used to generate a sequence logo in order to gain insight into variable or conserved regions. We have included this analysis in a figure below (Author response image 1).

Author response image 1

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We have also included a reference image equal to Figure 1b but with numbering of the extracellular loops to orient the reader. The mutations observed in V. cholerae isolates in this study, and experimentally validated to confer ICP2 resistance, are highlighted in yellow. The results of this analysis indicate that there is significant variability in OmpU, specifically in the predicted extracellular loops. As the reviewers point out, there are other V. cholerae isolates in GenBank that share the mutations identified in this study. There are also highly variable loops (i.e., loops 2 and 6) that we did not observe mutations in our data set, and it is not known if variability in these loops can confer phage resistance.

The strains included in the analysis below represent a broad collection of both environmental and clinical V. cholerae isolates from geographically disparate locations. Trying to identify the source of these deposited sequences is not always possible. Since OmpU represents an abundant outer membrane protein it is not surprising to observe this variability, which as the reviewers point out, is likely due to many different causes. One could imagine that the variability may be due to immune pressure, or phage pressure from ICP2 and/or other uncharacterized phages. To-date ICP2 like phages have only been isolated from clinical samples in Haiti and Bangladesh and we do not know if they co-exist with environmental non-toxigenic V. cholerae in these locations, or for that matter anywhere else in the world. We feel that the analysis provided in this manuscript which is the analysis of over 50 clinical strains in Bangladesh collected over a 10 year period, which share a niche with ICP2, is particularly relevant to making conclusions about the selective force exerted by ICP2 over time. In addition, we were able to experimentally confirm our hypothesis that these mutations confer ICP2 resistance.

Author details

1. Kimberley D Seed

   Department of Molecular Biology and Microbiology, Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, United States

   Contribution

   KDS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Minmin Yen

   Department of Molecular Biology and Microbiology, Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, United States

   Contribution

   MY, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. B Jesse Shapiro

   Département de sciences biologiques, Université de Montréal, Montreal, Canada

   Contribution

   BJS, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Isabelle J Hilaire

   Partners In Health, Boston, United States

   Contribution

   IJH, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Richelle C Charles

   1. Division of Infectious Diseases, Massachusetts General Hospital, Boston, United States
   2. Department of Medicine, Harvard Medical School, Boston, United States

   Contribution

   RCC, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

6. Jessica E Teng

   1. Partners In Health, Boston, United States
   2. Division of Global Health Equity, Brigham and Women's Hospital, Boston, United States

   Contribution

   JET, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Louise C Ivers

   1. Partners In Health, Boston, United States
   2. Division of Global Health Equity, Brigham and Women's Hospital, Boston, United States
   3. Department of Global Health and Social Medicine, Harvard Medical School, Boston, United States

   Contribution

   LCI, Conception and design, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Jacques Boncy

   National Public Health Laboratory, Port-au-Prince, Haiti

   Contribution

   JB, Conception and design, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

9. Jason B Harris

   1. Division of Infectious Diseases, Massachusetts General Hospital, Boston, United States
   2. Department of Medicine, Harvard Medical School, Boston, United States
   3. Department of Pediatrics, Harvard Medical School, Boston, United States

   Contribution

   JBH, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

10. Andrew Camilli

    Department of Molecular Biology and Microbiology, Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, United States

    Contribution

    AC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    andrew.camilli@tufts.edu

    Competing interests

    The authors declare that no competing interests exist.

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